a. Field of the Invention
The instant invention relates to spectroscopic assays and tagging.
b. Background
Spectroscopy is often used to analyze samples for their chemical composition. In some cases the sample is pure enough or has a high enough concentration of the material of interest that spectroscopic analysis can be performed directly on the sample. However, it is most often the case that a sample is very impure and the impurities will interfere with the spectroscopic analysis or the constituent that is desired to be detected is at a very low concentration; below the detection limit. The combination of impure sample and low concentration is also common and leads to the most challenging type of spectroscopic analysis.
Some examples of difficult analyses are: water analysis for trace contaminants or pathogens, where the sample may have some degree of purity; or analysis of biological sample where the sample matrix is very complex and the analyte is found at low concentrations. Often in these situations the samples are modified by filtering or a separation technique to enhance the concentration of the analyte or to remove the interfering material from the matrix. These additional steps often require time and expense. It would be desirable to have an analytical technique (assay) that is capable of performing all of these tasks rapidly and at low cost.
In 1999 a paper was published which showed how one could detect trace materials in complex matrices. This paper focused on the detection and quantitation of a metabolite, bilirubin, in biological samples. Bilirubin concentrations in blood or urine are desired as they indicate potential problems with liver function. It is of particular value with neonatal infants who are breaking down their mother's blood to bilirubin before their livers are functioning well enough to remove the toxic material.
In this publication the authors showed how a spectroscopic method known as Surface Enhanced Raman Scattering (SERS) could be used to detect bilirubin which has reacted with a synthetic coating on silver nanoparticles. The coating was synthesized to be highly reactive to the bilirubin molecule. The concept behind this analysis is to place the highly reactive coated SERS into a sample (whole blood) containing metabolic levels of bilirubin and to detect reaction product. The coating is illustrated in FIG. 1.
The authors were able to demonstrate that a small spectral feature was observed due to the reaction product and that peak could be used for quantitation. The spectra and resulting quantitative response curve are illustrated in FIG. 2. Even though the SERS effect enhances the spectral signal by as much as 108 the spectrum shown in FIG. 2 is dominated by features from whole blood components in the matrix. This interference greatly limits the quantitation of bilirubin in whole blood and prevents the detection of low quantities.
Another limitation to the detection limit of material is the particle nature of SERS. SERS requires nanoparticles of certain metals to be on the order of 100 nm. As the amount of analyte decreases it is necessary to concomitantly decrease the number of particles such that the amount of reacted analyte on the surface is high. Or conversely, the equivalent problem is that at low concentration the number of particles with a reacted bilirubin on the surface becomes small compared with those without a bilirubin. In either case, this situation is illustrated in FIG. 3. The sample will contain many particles that are not in the laser beam, and therefore, they are not detected.
This limit is fundamental. One cannot simply use a larger laser beam to interrogate more particles. The size of the laser beam is related to the spectral resolution. This is illustrated in FIG. 4. A spectroscopic analyzer, in the illustration we show the common Czerny-Turner design, produces a spectral resolution that is related to several factors, one of which is the aperture width, w. As w becomes larger the spectral resolution becomes worse. The spectral resolution must be sufficient to resolve the analyte spectral feature from the matrix features. The spectral resolution also is a factor in the detection limit as the height of the peak (known as the signal) is decreased with the loss of spectral resolution. This fundamental limit will limit the sensitivity of an assay even in pristine samples.
A solution to this fundamental limit was published as a patent application in 2006 (U.S. patent application Ser. No. 11/211,325) by Canon and Ray. Their method is illustrated in FIG. 5. FIG. 5A illustrates the concept of their paramagnetic pull-down assay. Two particles are used for this assay: a paramagnetic particle and a SERS active nanoparticle. Both particles are modified. The paramagnetic particle is modified with an analyte binding coating. The SERS active nanoparticle is coated with a material that produces a strong Raman signal, a protective coating, such as a SiO2 coating, and an analyte binding coating. The protective coating protects the SERS active nanoparticle and provides a surface to bind the analyte binding coating.
Figure illustrates the result of a positive paramagnetic pull-down assay. The assay results in a “sandwich” composed of a paramagnetic particle, the analyte, and a SERS active nanoparticle. This sandwich will produce a large SERS signal due to the coated SERS active nanoparticle. However, at this step of the assay the sample will always exhibit a large SERS signal since the SERS active nanoparticles are always present. Further, the signal produced will be that of the coating and is not related to the analyte.
FIG. 6 shows steps of a complete paramagnetic pull-down assay 10. The assay begins by adding a sample to a pre-treated vial in operation 12. The pre-treated vial contains the two particle types described in FIG. 5. The particle types may, for example, be in the form of a pellet or a coating on the inside of the vial. The sample is added to the vial in operation 14, and the vial is shaken vigorously to disrupt the particles and mix the particles in a solution with the sample in operation 16. The vial is then gently shaken to ensure that the particles stay suspended and convectively mix with the sample in operation 18. The final step uses a magnet to pull the paramagnetic material to a point in operation 20.
FIG. 7 demonstrates the significance of an invention described in 2006 U.S. patent application Ser. No. 11/211,325 by Carron and Ray (the '325 application), which is incorporated herein by reference in its entirety. In a system described in the '325 application, the pellet produced by a magnetic pull-down will only contain SERS active particles if the analyte is present to create the sandwich shown in FIG. 5B. The pull-down removes the particles from the sample matrix to effectively remove interferences from the matrix and it allows a small laser beam to interrogate all of the assay's SERS active particles that are indicative of a positive result. This is illustrated in FIG. 7A.
FIG. 7B shows that one could use the sample vial as an internal standard to normalize differences between sample vials or instrumentation to make a globally valid assay. FIG. 7C shows a result of the assay—a calibration curve with accurate predicted concentrations of analyte and a detection limit described by the RDL (Reliable Detection Limit).
If, as shown in FIG. 6, the pre-treated vial contains particles with a plurality of analyte binding coatings and an associated unique Raman coating on the SERS active particles for each analyte specific coating, a multiplex assay is created. A multiplex assay has the advantage of testing for multiple analytes simultaneously. For example, a medical assay for sexually transmitted diseases may contain the particle elements needed to detect several diseases at one time.